Team:Paris Saclay/Notebook/July/30
From 2013.igem.org
(Difference between revisions)
Line 176: | Line 176: | ||
Used quantities : | Used quantities : | ||
+ | * Oligo 43F : 0.5µL | ||
+ | * Oligo 44R : 0.5µL | ||
+ | * Buffer Dream Taq : 35µL | ||
+ | * DNA : 2µL | ||
+ | * dNTP : 0.5µL | ||
+ | * Dream Taq : ... cf cahier !!!!!!! | ||
+ | * H2O : ... cf cahier !!!!! | ||
+ | ===='''2 - Electrophoresis of the Colony PCR of Bba_K1155002 in DH5α'''==== | ||
+ | |||
+ | Abdou, Xavier | ||
+ | |||
+ | {| | ||
+ | | style="width:350px;border:1px solid black;" |File:Ps.jpg|350px]] | ||
+ | | style="width:350px;border:1px solid black;vertical-align:top;" | | ||
+ | * Well 1 : 6µL of DNA Ladder | ||
+ | * Well 2 : 5µL of PSB1C3+1µl of 6X loading dye | ||
+ | * Well 3 : 5µL of PSB1C3+1µl of 6X loading dye | ||
+ | * Well 4 : 5µL of BphR2 Part I+1µl of 6X loading dye | ||
+ | * Well 5 : 5 µL of BphR2 Part II+1µl of 6X loading dye | ||
+ | * Well 6 : 5µL of RBS-BphR2 Part I+1µl of 6X loading dye | ||
+ | * Well 7 : 5µL of FNR Part I+1µl of 6X loading dye | ||
+ | * Well 8 : 5µL of FNR Part II+1µl of 6X loading dye | ||
+ | * Well 9 : 5µL of RBS-FNR Part I+1µl of 6X loading dye | ||
+ | * Gel : 0.8% | ||
+ | |} | ||
{{Team:Paris_Saclay/incl_fin}} | {{Team:Paris_Saclay/incl_fin}} |
Revision as of 19:41, 15 September 2013
Notebook : July 30
Lab work
A - Aerobic/Anaerobic regulation system
Objective : obtaining Bba_K1155004, Bba_K1155005, Bba_K1155006
1 - Denaturation of EcoRI/SpeI used of the digestion of PCR products : NarK, NarG, NirB
Xavier
Protocol : Ethanol precipitation
We used 30µL of DNA.
Objective : obtaining Bba_K1155003
1 - Tranformation of RBS-AmilCP in PSB1C3 in DH5α
Abdou
Protocol : Bacterial transformation
A - Aerobic/Anaerobic regulation system / B - PCB sensor system
Objective : obtaining FNR and BphR2 proteins (Gibson assembly)
1 - PCR of : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I
Abdou, Xavier
Used quantities :
- Bphr2 Part I :
- Oligo 54F : 4 µL
- Oligo 55R :4 µL
- Buffer Dream Taq : 40 µL
- DNA : 2 µL
- dNTP : 4 µL
- Dream Taq : 1 µL
- H2O : 146 µL
- Bphr2 Part II :
- Oligo 54F : 4 µL
- Oligo 55R :4 µL
- Buffer Dream Taq : 40 µL
- DNA : 2 µL
- dNTP : 4 µL
- Dream Taq : 1 µL
- H2O : 146 µL
- RBS-Bphr2 Part I :
- Oligo 54F : 4 µL
- Oligo 55R :4 µL
- Buffer Dream Taq : 40 µL
- DNA : 2 µL
- dNTP : 4 µL
- Dream Taq : 1 µL
- H2O : 146 µL
- FNR Part I :
- Oligo ... : 4 µL
- Oligo ... :4 µL
- Buffer Dream Taq : 40 µL
- DNA : 2 µL
- dNTP : 4 µL
- Dream taq : 1 µL
- H2O : 146 µL
- FNR Part II :
- Oligo ... : 4 µL
- Oligo ... :4 µL
- Buffer Dream Taq : 40 µL
- DNA : 2 µL
- dNTP : 4 µL
- Dream Taq : 1 µL
- H2O : 146 µL
- RBS-FNR Part I :
- Oligo ... : 4 µL
- Oligo ... :4 µL
- Buffer Dream Taq : 40 µL
- DNA : 2 µL
- dNTP : 4 µL
- Dream Taq : 1 µL
- H2O : 146 µL
- PSB1C3 :
- Prefixe ... : 4 µL
- Suffixe ... :4 µL
- Buffer Dream Taq : 40 µL
- DNA : 2 µL
- dNTP : 4 µL
- Dream Taq : 1 µL
- H2O : 146 µL
PCR Program :
- BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I :
- FNR Part I, FNR Part II, RBS-FNR Part I :
- PSB1C3 :
2 - Electrophoresis of the PCR products : BphR2 Part I, BphR2 Part II, RBS-BphR2 Part I, FNR Part I, FNR Part II, RBS-FNR Part I
File:Ps.jpg|350px]] |
|
Expected size
- BphR2 Part I : 178 kb
- BphR2 Part II : 790 kb
- RBS-BphR2 Part I : 197 kb
- FNR Part I : 597 kb
- FNR Part II : 200 kb
- RBS-FNR Part I : 615 kb
- PSB1C3 : ...
We can't see RBS-BphR2 Part I fragments at the good size. We will make the PCR again with a new PCR Program. We obtain BphR2 Part I, BphR2 Part II, FNR Part I, Fnr Part II and RBS-FNR Part I frangments at the right size thanks to the PCR. We will purify it. |
3 - PCR of : RBS-BphR2 Part I
Abdou, Xavier
Used quantities :
- Oligo 54F : 4 µL
- Oligo 55R :4 µL
- Buffer .... : 40 µL
- DNA : 2 µL
- dNTP : 4 µL
- .... : 1 µL
- H2O : 146 µL
PCR Program :
4 - Gel purification of PCR of : RBS-BphR2 Part I
????????
B - PCB sensor system
Objective : obtaining Bba_K1155002
1 - Colony PCR of Bba_K1155002 in DH5α
Abdou, Xavier
Used quantities :
- Oligo 43F : 0.5µL
- Oligo 44R : 0.5µL
- Buffer Dream Taq : 35µL
- DNA : 2µL
- dNTP : 0.5µL
- Dream Taq : ... cf cahier !!!!!!!
- H2O : ... cf cahier !!!!!
2 - Electrophoresis of the Colony PCR of Bba_K1155002 in DH5α
Abdou, Xavier
File:Ps.jpg|350px]] |
|